Computational Prediction for Antibiotics Resistance Through Machine Learning and Pk/Pd Analysis by Hyunjo Kim in Cohesive Journal of Microbiology & Infectious Disease

1. Computational Prediction for Antibiotics
Resistance Through Machine Learning and Pk/Pd
Analysis
Hyunjo Kim1
* and Jaehoon Song2
1
School of pharmacy, Yonsei university, South Korea
2
ANSORP, CHA Bio Group at CHA Bio complex, South Korea
Introduction
Analarmingandpersistentriseinantibioticresistanceamongmanyimportantpathogenic
bacterial species poses one of the greatest contemporary challenges to the public health. The
treatment of bacterial infections is becoming increasingly ineffective due to rapid mutation
which leads to antibiotic resistant and resistant bacteria become more prevalent. As result the
existing antibiotics are gradually obsolete and again new drugs are needed to be designed for
the same threat. Recently, antibiotic resistance is a global health crisis linked to increased, and
often unrestricted, antibiotic use in humans and animals [1]. The rise of multiclass-antibiotics
resistant pathogens and the dearth of new antibiotic development place an existential strain
on successful infectious disease therapy. Breakthrough strategies that go beyond classical
antibiotic mechanisms are needed to combat this looming public health catastrophe.
Reconceptualizing antibiotic therapy in the richer context of the host-pathogen interaction is
required for innovative solutions. Here we review these relationships and their relevance to
antimicrobial resistance (AMR) trends witnessed in the clinical setting. This review highlights
the issues of enrichment and dissemination of antibiotics resistance genes (ARGs) [2,3] in
the environment, and also future needs in mitigating the spread of antibiotic resistance
in the environment, particularly under the planetary health perspective, i.e., the systems
that sustain or threaten human health. By defining specific virulence factors, the essence
of a pathogen, and pharmacologically neutralizing their activities, one can block disease
progression and sensitize microbes to immune clearance. Likewise, host-directed strategies
to boost phagocyte bactericidal activity, enhance leukocyte recruitment, or reverse pathogen-
induced immunosuppression seek to replicate the success of cancer immunotherapy in the
field of infectious diseases. The answer to the threat of multidrug-resistant pathogens lies in
current antibiotic paradigms. Furthermore, the rise of bacterial strains resistant to multiple
antibiotics is expected to dramatically limit treatment effectiveness, leading to potentially
incurable outbreaks [4]. In addition to new drug development efforts, there is an urgent need
for preclinical tools that are capable of effective and rapid detection of resistance, as culture-
based laboratory diagnostics test are usually time consuming and costly. One of the major
Crimson Publishers
Wings to the Research
Review Article
*1
Corresponding author: Hyunjo Kim,
School of pharmacy, Yonsei university,
Campus town, Songdo techno park, Incheon
city, South Korea
Submission: June 13, 2019
Published: June 24, 2019
Volume 2 - Issue 5
Howtocitethisarticle:HyunjoK, Jaehoon
S. Growth Factors in the Human Body: A
Conceptual Update. Cohesive J Microbiol
Infect Dis. 2(5). CJMI.000547.2019.
DOI: 10.31031/CJMI.2019.02.000547
Copyright@ Hyunjo Kim, This article is
distributed under the terms of the Creative
Commons Attribution 4.0 International
License, which permits unrestricted use
and redistribution provided that the
original author and source are credited.
ISSN: 2578-0190
1
Cohesive Journal of Microbiology & Infectious Disease
Abstract
Infection disease is a major cause of morbidity and mortality in the developing world. Antibiotics
resistance, which is predicted to rise in many countries worldwide, threatens infection treatment and
control. The machine learning can help to identify patients at higher risk of treatment failure in infection
diseases Closer monitoring of these patients may decrease treatment failure rates and prevent emergence
outbreak of antibiotic resistance. To identify features associated with treatment failure and to predict
which patients are at highest risk of antibiotics resistance this study was designed. The most predictive
model was forward stepwise selection, although most models performed at or above targeted value. We
employed a range of powerful machine learning tools to predict antibiotic resistance from whole genome
sequencing data for model strain. We used the presence or absence of genes, population structure and
isolation year of isolates as predictors, and could attain average precision and recall without prior
knowledge about the causal mechanisms. These results demonstrate the potential application of machine
learning methods as a diagnostic tool in healthcare settings.
Keywords: Infection disease; Antibiotics resistance; Machine learning model algorithm; Genome
analysis; Antibiotics resistance genes (ARGs); PK/PD analysis

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health threats of 2st
century is emergence of antibiotic resistance.
To manage its human health and economic impact, efforts are
made to develop novel diagnostic tools that rapidly detect resistant
strains in clinical settings [5,6]. In our study, we employed a range
of powerful machine learning tools to predict antibiotic resistance
from whole genome sequencing data for model bacteria. We used
the designed machine learning algorithms as predictors, and
could train average precision of the aimed value and recall of the
closest number to mean without prior knowledge about the causal
mechanisms considering environmental factors [7-9] related to
key parameters such as antibiotic resistomes [10-14] and their
mechanisms [15,16]. These results demonstrate the potential
application of machine learning methods as a diagnostic tool in
healthcare settings. Therefore, systemically designed algorithms
based strategies to mitigate the spread of antibiotic resistance are
suggested [17,18].
Overall, this article provides a conceptual framework for
understanding the complexity of the problem of emergence of
antibiotic resistance in the clinic. Availability of such knowledge will
allow researchers to build models for dissemination of resistance
genes and for developing interventions to prevent recruitment of
additional or novel genes into pathogens.
Investigational Background
The successful treatment of infectious diseases heavily relies
on the therapeutic usage of antibiotics. However, the high use of
antibiotics in humans and animals leads to increasing pressure
on bacterial populations in favorite of resistant phenotypes.
Antibiotics reach the environment from a variety of emission
sources and are being detected at relatively low concentrations.
Given the possibility of selective pressure to occur at sub-inhibitory
concentrations, the ecological impact of environmental antibiotic
levels on microbial communities and resistance levels is vastly
unknown. Quantification of antibiotics resistance genes (ARGs) and
of antibiotic concentrations is becoming commonplace. Yet, these
two parameters are often assessed separately and in a specific
spatiotemporal context, thus missing the opportunity to investigate
how antibiotics and ARGs relate. Furthermore, antibiotics (multi)
resistance has been receiving ever growing attention from
researchers, policy-makers, businesses and civil society. Our aim
wastocollectthelimiteddataonantibioticconcentrationsandARGs
abundance currently available to explore if a relationship could
be defined. A metric of antibiotic selective pressure, i.e. the sum
of concentrations corrected for microbial inhibition potency, was
used to correlate the presence of antibiotics in the environment to
total relative abundance of ARGs while controlling for basic sources
of non-independent variability, such as strain and antibiotic class.
The results of this meta-analysis show a significant statistical effect
of antibiotic pressure and type of environmental compartment on
the increase of ARGs abundance even at very low levels. Moreover,
our analysis emphasizes the importance of integrating existing
information particularly when attempting to describe complex
relationships with limited mechanistic understanding in our
previous paper (Figure 1).
Figure 1: Schematic illustration of antibiotics resistance investigation.
Hence, the application of metagenomic functional selections to
study antibiotics resistance genes is revealing a highly diverse and
complex network of genetic exchange between bacterial pathogens
and environmental reservoirs, which likely contributes significantly
to increasing resistance levels in pathogens. In some cases, clinically
relevant resistance genes have been acquired from organisms
where their native function is not antibiotics resistance, and which
may not even confer a resistance phenotype in their native context.
In this review, we attempt to distinguish the resistance phenotype
from the resistomes genotype, and we highlight examples of genes
andtheirhostswherethisdistinctionbecomesimportantinorderto
understand the relevance of environmental niches that contribute
most to clinical problems associated with antibiotic resistance.

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Methods
We conduct a systematic review, selecting studies if they are
published randomized controlled trials (RCTs) which report the
relationship between taking any antibiotics for any indication
and incidence of resistant Strains in patients of any age group.
We use a predefined search strategy to identify studies meeting
these eligibility criteria in MEDLINE, Embase, Global Health and
the Cochrane Central Register of RCTs. Two authors independently
screen titles and abstracts, review the full texts and undertake data
extraction. If feasible, we will perform pair-wise meta-analysis
modelling to determine the relationship between the duration
of antibiotics treatment and development of resistant Strain. If
the identified studies meet the assumptions for a network meta-
analysis, we additionally model this relationship using indirect
comparisons. This hypothesis illustrated in Figure 2 as an example
for macrolide class and its hypothetical network of anticipated
randomized controlled trial data for the effect of macrolide
treatment duration on the development of antimicrobial resistance
is presented. As shown in Figure 2, each treatment group is a node.
The lines joining nodes, termed edges, will be drawn to thickness
that graphically represents the amount of direct evidence: the
number of comparisons that we expect to find between a particular
pair of nodes.
Figure 2: Hypothetical network of anticipated randomised controlled trial data for the effect of chosen
antibiotics treatment duration on the development of antimicrobial resistance.
Mechanisms of antibiotic resistance: genetic basis
Emergence of resistance among the most important bacterial
pathogens is recognized as a major public health threat affecting
humans worldwide [19-22]. Multidrug-resistant organisms have
not only emerged in the hospital environment but are now often
identified in community settings, suggesting that reservoirs of
antibiotics resistant bacteria are present outside the hospital [23-
25]. The bacterial response to the antibiotic attack is the prime
example of bacterial adaptation and the pinnacle of evolution.
Survival of the fittest is a consequence of an immense genetic
plasticity of bacterial pathogens that trigger specific responses that
result in mutational adaptations, acquisition of genetic material, or
alteration of gene expression producing resistance to virtually all
antibioticscurrentlyavailableinclinicalpractice[26-28].Therefore,
understanding the biochemical and genetic basis of resistance is of
paramount importance to design strategies to curtail the emergence
and spread of resistance and to devise innovative therapeutic
approaches against multi-antibiotics resistant organisms.
Overview on evolutionary processes of the antibiotic resistance
Figure 3: Overview evolutionary processes of the antibiotic resistance.

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During millions of years antibiotics and antibiotic resistance
genes have co-evolved slowly. In this long period the first transition
was the acquisition of pre-resistance genes by different bacteria.
This genetic transference allowed the evolution toward true and
more efficient antibiotic resistance genes. However, the great
evolutionary transition was the discovery, mass production and
consumption of antibiotics. Antibiotics accelerated dramatically
the diversification of resistance genes and selection for reaching
extraordinary efficient variants (Figure 3). In this study, we will
describe in detail the major mechanisms of antibiotic resistance
encountered in clinical practice, providing specific examples in
relevant bacterial pathogens (Table 1).
Table 1: Different examples of resistance mechanisms
with clinical relevance from natural functions in environ-
mental bacteria.
Antimicrobial
classified
group
Resistance
Mechanisms
Related Natu-
ral Protein
Natural Reser-
voirs
Antibiotics
Affected
Aminoglyco-
sides
Acetylation
Phosphoryla-
tion
Histone-acety-
lases Protein
kinases
Streptomyces
Tetracyclines Efflux (mar)
Major facilita-
tor superfamily
EF-Tu, EF-G
Streptomyces
Chloramphen-
icol
Acetylation
Efflux (mar)
Acetylases Ma-
jor facilitator
superfamily
EF-Tu, EF-G
Streptomyces
Macrolides Target mutation
50S ribosomal
subunit
Streptomyces
β-lactams
(methicillin)
PBP2a
Homologous
PBP2a
Staphylococcus
sciuri
β-lactams (car-
bapenems)
OXA-48 inacti-
vating enzyme
Proteins
participating in
peptidoglycan
synthesis
Shewanella xia-
menensis
OXA-23 inacti-
vating enzyme
Proteins
participating in
peptidoglycan
synthesis
Acinetobacter
radioresistens
Inactivating
process
L1 beta-lact-
amase
Stenotrophomon-
as maltophilia
Cephalospo-
rins
Inadequate
target
PBP5 muta-
tions
Enterococcus
faecium
Fluoroquino-
lones
Topoisomerase
protection
Qnr-like pro-
tein
Shewanella algae
Nowadays, the high-throughput sequencing tools and
bioinformatics software, knowledge on high bacterial diversity in
bacterialcommunities(metagenome)isincreasing.Ahugediversity
of resistance mechanisms to practically all antibiotic families
has been found in both antibiotic- and non-antibiotic-producing
bacteria. Three types of resistomes can be defined: intrinsic,
environmental, and unknown [29-33]. In the intrinsic resistome or
pre-resistome, the antibiotic resistant elements belong to bacterial
metabolic networks, reflecting their role in microbial physiology.
They might be coupled to signaling molecules (antibiotics)
facilitating the co-selection of antibiotics and antibiotic resistance
genes in a constant arms-race over a long time. The intrinsic
resistome is a wider concept and probably universal to the bacterial
world. The description of the intrinsic resistome has expanded our
knowledge about potential new resistance mechanisms [34-36].
They could become true antibiotic resistance genes if appropriate
driving forces were exerted. Moreover, the potential adaptiveness
of these pre-resistome genes can be accelerated if, by chance, they
are transferred to new genetic contexts (Figure 3), where these
genes may evolve toward more efficient enzymes without having a
physiological role. As a consequence, this silent and non-predictive
resistome (unknown resistome) is ready to be selected [37-39].
Mutational resistance gene transfer
Figure 4: Schematic representation of the mechanism of action and resistance to linezolid. Panel A. Linezolid
interferes with the positioning of aminoacyl-tRNA; Panel B. Representation of domain V of 23S rRNA.

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Bacteria have a remarkable genetic plasticity that allows them
to respond to a wide array of environmental threats, including the
presence of antibiotic molecules that may jeopardize their existence
[40,41]. As mentioned, bacteria sharing the same ecological niche
with antimicrobial-producing organisms have evolved ancient
mechanisms to withstand the effect of the harmful antibiotic
molecule and, consequently, their intrinsic resistance permits them
to thrive in its presence [42,43]. From an evolutionary perspective,
bacteria use two major genetic strategies to adapt to the antibiotic
attack, mutations in gene(s) often associated with the mechanism of
action of the compound, and acquisition of foreign DNA coding for
resistance determinants through horizontal gene transfer [44,45].
In this scenario, a subset of bacterial cells derived from a
susceptible population develop mutations in genes that affect
the activity of the drug, resulting in preserved cell survival in the
presence of the antimicrobial molecule. Once a resistant mutant
emerges, the antibiotic eliminates the susceptible population and
the resistant bacteria predominate. In many instances, mutational
changes leading to resistance are costly to cell homeostasis and
are only maintained if needed in the presence of the antibiotic. In
general, mutations resulting in antimicrobial resistance alter the
antibiotic action via one of the following mechanisms (Figure 4).
Mutation of target sites
One of the most efficient mechanisms for accumulating
antimicrobial resistance genes is represented by integrons, which
are site-specific recombination systems capable of recruiting open
reading frames in the form of mobile gene cassettes. Integrons
provide an efficient and rather simple mechanism for the addition
of new genes into bacterial chromosomes, along with the necessary
machinery to ensure their expression; a robust strategy of genetic
interchange and one of the main drivers of bacterial evolution.
Thus, resistance arising due to acquired mutational changes is
diverse and varies in complexity [46-50]. Classically, bacteria
acquire external genetic material through three main strategies,
transformation (incorporation of naked DNA), transduction (phage
mediated) and, conjugation. Emergence of resistance in the hospital
environment often involves conjugation, a very efficient method of
gene transfer that involves cell-to-cell contact and is likely to occur
at high rates in the gastrointestinal tract of humans under antibiotic
treatment. As a general rule, conjugation uses mobile genetic
elements (MGEs) as vehicles to share valuable genetic information,
although direct transfer from chromosome to chromosome has
also been well characterized. The most important MGEs are
plasmids and transposons, both of which play a crucial role in the
development and dissemination of antimicrobial resistance among
clinically relevant organisms. In other words, antibiotics resistance
mechanisms are summarized as following; modifications of the
antimicrobial target (decreasing the affinity for the antibiotics),
a decrease in the drug uptake, activation of efflux mechanisms
to extrude the harmful molecule, or global changes in important
metabolic pathways via modulation of regulatory networks [51].
For an instance, the ErmC leader peptide (Figure 5) is produced
and the ermC mRNA forms two hairpins, preventing the ribosome
to recognize the ribosomal binding site (RBS) of ermC. As a result,
translation is inhibited under non-induction conditions. After
exposuretoerythromycin,theantibioticinteractswiththeribosome
and binds tightly to the leader peptide, stalling progression of
translation. This phenomenon releases the ermC RBS and permits
translation.
Figure 5: Schematic representation of the post-transcriptional control of the gene. RBSL, ribosomal(blue)
binding site of the leader; RBSC, ribosomal binding site of ermC; AUG, initiation codon.
Therefore, bacteria have evolved a sophisticated mRNA-based
control mechanism to tightly regulate the expression of these
methylases, ensuring a high efficiency of action in the presence of
the antibiotic while minimizing the fitness costs for the bacterial
population. Similarly, the system is usually not induced by
lincosamides [46,47] or streptogramins [48]. However, the use of
these agents against isolates carrying inducible erm genes may
result in the selection of constitutive mutants in vivo (particularly
in severe infections), leading to therapeutic failures.

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Prediction of antibiotic resistance
The emergence of microbial antibiotic resistance is a global
health threat. In clinical settings, the key to controlling spread of
resistant strains is accurate and rapid detection. As traditional
culture-based methods are time consuming, genetic approaches
have recently been developed for this task. The detection of
antibiotics resistance is typically made by measuring a few known
determinants previously identified from genome sequencing, and
thus requires the prior knowledge of its biological mechanisms. To
overcome this limitation, we employed machine learning models
to predict resistance to chosen compounds across multi-classes
of antibiotics from existing and novel whole genome sequences
of model strains [17]. It was considered a range of methods, and
examined population structure, isolation year, gene content,
and polymorphism information as predictors. Gradient boosted
decision trees consistently outperformed alternative models
with an average accuracy of the aimed value on held-out data.
While the best models most frequently employed gene content,
an average accuracy scores could be obtained using population
structure information alone. Single nucleotide variation data were
less useful, and significantly improved prediction only for chosen
antibiotics and its combination through given model algorithms
through modification specially. These results demonstrate that
antibiotics resistance can be accurately predicted from whole
genome sequences without a strain knowledge of mechanisms, and
that both genomic and epidemiological data can be informative.
This paves way to integrating machine learning approaches
into diagnostic tools in the clinic [18]. Still, the understanding of
mode of evolution of resistance in bacteria is a determining step
in the preclinical development of new antibiotics, because drug
developers assess the risk of resistance arising against a drug
during preclinical development.
Antibiotics resistance measurements index
The aim of the study was to evaluate a cumulative antibiotics
resistance index (ARI) as a possible key outcome measure of
antimicrobial stewardship programs and as a tool to predict
antimicrobial resistance trend. Antibiotic susceptibility for model
strains pathogens, recovered from blood cultures during a fixed
time period, was analyzed to obtain a cumulative ARI. For each
antibiotic tested a score of 0, 0.25, 0.5, 0.75 or 1 was assigned for
susceptibility, intermediate resistance, or resistance, respectively,
and the ARI was calculated by dividing the sum of these scores by
the number of antibiotics tested. Cumulative ARI of microorganisms
were compared, and a mathematical prediction model for
antimicrobial resistance trend was obtained. ARI could be a useful
tool to measure the impact of antibiotics surveillance programs
on antibiotics resistance. This clinical prediction rule performed
well with internal validation. It showed good calibration and good
discrimination. The receiver operating curve is shown in Figure 6.
Figure 6: Prediction model AUC.
Predicting antibiotic resistance from resistance genes.
It has been developed a high-throughput multiplex PCR test
for several resistance genes encoding OpGen. OpGen offers the
high-throughput multiplex PCR test as a commercial testing
service for analysis of bacterial isolates. The resistance genes were
chosen based on a review of the scientific literature and surveys of
resistance gene databases. PCR assays specific for gene variants at
the amino acid positions indicated or homologous sequence regions
shared by different gene subtypes within a family of closely related
resistance genes were designed in Table 2. The resistance genes
in database effectively covered the resistance mechanisms for the
isolates in this study and could be used for the sensitive prediction
of phenotypic resistance with generalized linear models, which
allowed the inference of phenotypic susceptibility in the absence
of predicted resistance. The binary models effectively predicted
phenotypic resistance based on the presence of resistance genes but
did not have sufficient sensitivity to infer phenotypic susceptibility
in the absence of resistance genes because the binary models lacked
full coverage of resistance mechanisms.

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Table 2: Percentage of isolates with non-susceptible phenotypes per antibiotic.
Antibiotic
% of Isolates
E. coli K. pneumoniae P. mirabilis P. aeruginosa
(n = 2,919) (n = 1,974) (n = 1,150) (n = 1,484)
Gentamicin 33 58 40 60
Tobramycin 46 76 38 51
Ciprofloxacin 68 81 58 67
Levofloxacin 67 72 47 69
Trimethoprim-sulfamethoxazole 58 75 63 Intrinsic resistance
Imipenem 7 39 Intrinsic resistance 64
Ertapenem 13 51 1 Intrinsic resistance
Cefazolin 74 90 49 Intrinsic resistance
Cefepime 68 84 29 69
Cefotaxime 71 86 41 Intrinsic resistance
Ceftazidime 55 84 22 62
Ceftriaxone 71 87 41 Intrinsic resistance
Ampicillin 88 Intrinsic resistance 72 Intrinsic resistance
Aztreonam 67 84 13 64
Additionally, genome retrieval and identification analyses
were performed. The complete genome of Strain sequences
was downloaded from The National Center for Biotechnology
Information (NCBI) Genome Database (https://www.ncbi.nlm.
nih.gov/genome). The fasta file format of the genome sequence
of 11 strains of bacteria were thoroughly analysed for Antibiotics
Resistance Genes (ARGs) on the bulk analysis Resistance Gene
Identifier (RGI) or CARD 2017 Platform (https://card.mcmaster.
ca/ analyze/rgi). Default select criteria, which identified gene base
on strict or perfect only was used. On the RGI platform each genome
sequence file was uploaded and all settings were left at default.
To have an inter-relation as well as qualitative and quantitative
pattern of these ARGs in the various strains, a heatmap chart was
constructed using Microsoft Excel 2016 version for Mac.
Mathematical modeling
All sequencing reads sets were used as inputs to each of
antibiotics resistance prediction algorithms. In case of no calls or
if there were discordant results among the reads sets of a sample
for antibiotics, the prediction for the sample for that particular
antibiotics was treated as missing. In our sensitivity and specificity
calculation and parameter estimation for DST [52,53] credibility
computation, which are subsequently described in detail, we
omitted those samples clearly indicated to have been used to train
the tools. The sensitivities and specificities of the algorithms for
antibiotics were first calculated together with their 95% confidence
interval, with the corresponding phenotypes in the data collection
as the gold standard. Bioinformatics tools have also been developed
to predict drug resistance from whole genome sequencing (WGS)
data. Sufficiently large databases possessing both WGS and DST
information have allowed the drug resistance to several antibiotics
to be accurately predicted, to the extent that rapid diagnostics that
target these mutations have been developed [54-57].
The DST credibility score for each sample was calculated as
the probability specifically, for antibiotics i, let the prevalence
of antibiotics resistance be Pi, and the true sensitivity, the true
specificity, the proportion of no-call predictions among truly
resistant samples and the proportion of no-call predictions among
truly susceptible samples of the program be Sensij, Specij, NoRij,
and NoSij, for j in {StrainProfiler, Anibiotics}. We denote the
predictions of the algorithms as
𝑝𝑟𝑒𝑑𝑖𝑐𝑡𝑖𝑜𝑛𝑠 =𝑌𝑗𝑗 ∈ {Strain𝑃𝑟𝑜𝑓𝑖𝑙𝑒𝑟, Antibiotics},
where Yj = 1 if the sample is predicted resistant, 0 if susceptible,
and NA otherwise. The probability of the sample being resistant
given the predictions of the four tools is as follows:
𝑃 (𝑠𝑎𝑚𝑝𝑙𝑒 𝑖𝑠 𝑟𝑒𝑠𝑖𝑠𝑡𝑎𝑛𝑡 | 𝑝𝑟𝑒𝑑𝑖𝑐𝑡𝑖𝑜𝑛𝑠) = 𝑃 (𝑝𝑟𝑒𝑑𝑖𝑐𝑡𝑖𝑜𝑛𝑠 |
𝑠𝑎𝑚𝑝𝑙𝑒 𝑖𝑠 𝑟𝑒𝑠𝑖𝑠𝑡𝑎𝑛𝑡) / 𝑃𝑖𝐿𝑖𝑘𝑒𝑙𝑖ℎ𝑜𝑜𝑑 (𝑝𝑟𝑒𝑑𝑖𝑐𝑡𝑖𝑜𝑛𝑠),
with Likelihood(predictions) calculated as
𝑃 (𝑝𝑟𝑒𝑑𝑖𝑐𝑡𝑖𝑜𝑛𝑠 | 𝑠𝑎𝑚𝑝𝑙𝑒 𝑖𝑠 𝑟𝑒𝑠𝑖𝑠𝑡𝑎𝑛𝑡).
𝑃𝑖 + 𝑃 (𝑝𝑟𝑒𝑑𝑖𝑐𝑡𝑖𝑜𝑛𝑠 | 𝑠𝑎𝑚𝑝𝑙𝑒 𝑖𝑠 𝑠𝑢𝑠𝑐𝑒𝑝𝑡𝑖𝑏𝑙𝑒).
(1−𝑃𝑖) = 𝑗𝑃 (𝑌𝑗 | 𝑠𝑎𝑚𝑝𝑙𝑒 𝑖𝑠 𝑟𝑒𝑠𝑖𝑠𝑡𝑎𝑛𝑡).
𝑃𝑖 + 𝑗𝑃 (𝑌𝑗 | 𝑠𝑎𝑚𝑝𝑙𝑒 𝑖𝑠 𝑠𝑢𝑠𝑐𝑒𝑝𝑡𝑖𝑏𝑙𝑒). (1−𝑃𝑖),
where
𝑃 (𝑌𝑗 | 𝑠𝑎𝑚𝑝𝑙𝑒 𝑖𝑠 𝑟𝑒𝑠𝑖𝑠𝑡𝑎𝑛𝑡) = {𝑆𝑒𝑛𝑠𝑖𝑗 𝑖𝑓 𝑌𝑗 = 1,
1−𝑆𝑒𝑛𝑠𝑖𝑗−𝑁𝑜𝑅𝑖𝑗 𝑖𝑓 𝑌𝑗=0,
𝑁𝑜𝑅𝑖𝑗 𝑖𝑓 𝑌𝑗 𝑖𝑠 𝑁𝐴,

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and
𝑃 (𝑌𝑗 ∣ 𝑠𝑎𝑚𝑝𝑙𝑒 𝑖𝑠 𝑠𝑢𝑠𝑐𝑒𝑝𝑡𝑖𝑏𝑙𝑒) = {1−𝑆𝑝𝑒𝑐𝑖𝑗−𝑁𝑜𝑆𝑖𝑗 𝑖𝑓 𝑌𝑗=1
𝑆𝑝𝑒𝑐𝑖𝑗 𝑖𝑓 𝑌𝑗=0
𝑁𝑜𝑆𝑖𝑗 𝑖𝑓 𝑌𝑗 𝑖𝑠 𝑁𝐴.
The DST credibility score of a sample thus equals P (sample
is resistant | predictions) if its reported phenotype is resistant, or
1 – P (sample is resistant | predictions) if the reported phenotype
is susceptible.
Prediction on antibiotic resistance at the PK/PD point
of view
Antibiotic resistance constitutes one of the most pressing
public health concerns [58-61]. Antimicrobial peptides (AMPs) of
multicellular organisms are considered part of a solution to this
problem, and AMPs produced by bacteria such as colistin are last-
resort drugs. Importantly, AMPs differ from many antibiotics in
their pharmacodynamic characteristics. Here we implement these
differences within a theoretical framework to predict the evolution
of resistance against AMPs and compare it to antibiotic resistance.
Our analysis of resistance evolution finds that pharmacodynamic
differences all combine to produce a much lower probability that
resistance will evolve against AMPs. The finding can be generalized
to all drugs with pharmacodynamics similar to AMPs [62-65].
Pharmacodynamic concepts are familiar to most practitioners
of medical microbiology, and data can be easily obtained for
any antibiotics or antibiotics combination. Our theoretical and
conceptual framework is, therefore, widely applicable and can help
avoid resistance evolution if implemented in antibiotic stewardship
schemes or the rational choice of new antibiotics candidates [66].
Mutant selection window and pharmacodynamic
parameters [67,68]
Schematically, the revised mutant selection window (MSW) and
pharmacodynamic parameters are shown in Figure 7. The MSW is
defined as the antimicrobial concentration range in which resistant
mutants are selected [69,70-71]. Following MSW using net growth
curves of a susceptible strain S and a resistant strain R are also
determined. Mathematically, net growth is described with the
pharmacodynamic function ψ(a). In short, the function consists of
the four pharamcodynamic parameters [67,68,72-74]: net growth
in the absence of antibicrobials ψ ax, net growth in the presence of
a dose of antimicrobials, which effects the growth maximal, ψmin,
the minimum inhibitory concentration (MIC) and the parameter
κ, which describes the steepness of the pharamcodynamic curve.
Here, the two pharmacodynamics functions ψS(a) and ψR(a)
describe the net growth of the S and R, respectively, in relation
to the drug concentration a. Cost of resistance c is included as
a reduction of the maximum growth rate of the resistant strain
ψmax,R, with c = 1-ψmax, R/ψmax,S. Note that with this definition,
cost of resistance is expressed as reduction in net growth rate in
the absence of antimicrobials (a = 0). The lower bound of the MSW
is the concentration for which the net growth rate of the resistant
strain is equal to the net growth rate of the sensitive strain and is
called the minimal selective concentration (MSC). The upper bound
is given by the MIC of the resistant strain MICR. It is calculated the
size of the MSW as: . Following the original approach to define
the MSW, the boundaries of the MSW can also be applied to the
pharmacokinetics of the system. The width of the MSW is partly
determined by the steepness of the pharmacodynamic curve
(Figure 7).
Figure 7: The mutant selection window (MSW) for arbitrary mutant strains.

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CJMI.000547. 2(5).2019
Mathematical modeling focused on Pharmacodynamics
To study resistance evolution, we used a mathematical
model [75,76] that incorporates pharmacokinetics (PK)/
pharmacodynamics (PD) [77] and captures population dynamics
of bacterial populations under treatment with antimicrobial
drugs. We ran stochastic simulations to calculate the probability
of resistance emergence, the probability of takeover by a resistant
strain, the time to resistance emergence and the risk of resistance.
Importantly, the concentration range between no killing and
maximal killing is much narrower for AMPs than antibiotics,
resulting in a much steeper curve. The maximum killing rate of
AMPs is much higher than of antibiotics, as reflected in quicker
killing time. These differences between AMPs and antibiotics with
respect to their pharmacodynamic parameters determine the size
of the MSW and enable us to assess the influence of the MSW on
resistance evolution. Another difference relevant to the evolution
of resistance is the finding that many antibiotics increase mutation
rates of bacteria, but the AMPs tested so far do not show such an
effect as they do not elicit bacteria DNA repair responses [78,79].
Here, we use a pharmacodynamics approach that has been
widely used to describe sigmoid dose-response relationships to
study the evolution of resistance in a homogeneous population.
Our work uses the formulation of pharmacodynamic function. We
particularly explored how the steepness of the pharmacodynamic
curve, together with other pharmacodynamic parameters
determine the probability of resistance emergence [80,81].
The size of the MSW depends on the lower and upper bound of
the MSW and is calculated as ratio, due to the logarithmic scale that
is used to plot dose–response relationships;
R
MSW
MIC
size
MSC
=
(1)
To analytically describe the MSW, we use the pharmacodynamic
function ψ(a), which mathematically describes the net growth rate
with a Hill function:
max min
max max
min max
( )( / )
( ) ( )
( / ) /
k
k
a MIC
a d a
a MIC
ψ ψ
ψ ψ ψ
ψ ψ
−
= − = −
−
(2)
The analytic solution of the MSC is
1/
,
min ,0 1
( 1)
max, min,
K
S
c S
MSC MIC c
s S c
S
ψ
ψ ψ
 
≤ ≤
 
− +
 
 
(3)
The population dynamics of the susceptible and resistant strains is
captured in the following system of differential equations:
[ ]
(1 ) 1
s S n
dS S R
r S d d S
dt K
µ
+
 
= − − − +
 
  (4)
and
[ ]
1 1
R s R n
dR S R S R
r R r S d d R
dt K K
µ
+ +
   
= − + − − +
   
   
To include the change of antimicrobial concentrations over time
(pharmacokinetics) into our model, we define the death rate to be
dependent on the time-dependent antimicrobial concentration
a(t):
( )
max min
min max
( ( ) / )
( ( )) , ,
( ( ) / ) /
K
i K
a t MIC
d a t i S R
a t MIC
ψ ψ
ψ ψ
−
= =
− (5)
We assume a time-dependent pharmacokinetic function a(t) of
the following form
[ ( 1) ] [ ( 1) ]
( ) ( ), 1,2,3.....
ke t n ka t n
a
n a e
Dk
a t e e n
k k
τ τ
− − − − − −
= − =
−
∑ (6)
We define the treatment dose as the average concentration
during the course of treatment:
1
( )
a a t dt
t
= ∫ (7)
Therefore, modeling hazard function can be written as
1
( , ) ( , ) ( ) ( )
R
H a t S a t ps R a a dt
Kt
ψ
= →
∫ (8)
In order to generate predictions on antibiotics resistance
based on pharmacodynamics, one of our main goals of the
project, we made a number of simplifying assumptions. The
pharmacodynamics are based on data of initial killing only.
Moreover, we assume homogeneous populations over time and
space. We implemented resistance, without considering whether
resistance mutations are costly or mitigated by compensatory
mutations. Our simulations suggest that resistance is limited in
predicting resistance evolution based on pharmacodynamics.
Expanding the framework to integrate tolerance and resistance
is possible but would require pharmacodynamic estimates and
additional functions. Another possible extension of our work would
be to include pharmacodynamic estimates of resistant strains that
change over time owing to compensatory mutations and to cross-
resistance or collateral sensitivity when exposed to combinations
of antimicrobials. Finally, we assumed the same pharmacokinetics
for all cases in our study. The future empirical work will inform
realistic parameter estimates for pharmacokinetics. In all cases,
however, the basis of any analysis concerning antibiotics resistance
is the influence of individual pharmacodynamic parameters, for
which we provide a framework.
Conclusion
Consequently, future studies should assess the value of even
broader data for accurate prediction, ranging from transcriptome
and proteome to other clinical and epidemiological data, such as
cross-resistance and history of antibiotic therapy. Integrating
these information sources from large isolate panels into a single
predictive framework will lead to a rational basis for introducing
machine learning in decision-making in public health.
Acknowledgement
I would like to appreciate great encouragement from Dr. Jae
Hoon, Song who is a chairman of ANSORP as well as CHA Bio Group
specially and this journal editor
Conflict of interest statement
Compliance with Ethical Standards The author reports no
conflict of interest.

10. 10
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CJMI.000547. 2(5).2019
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